1. Field of the Invention
This invention relates to a method for the rapid, simple and quantitative determination of a carbon source and a fermentation product in a culture medium by means of a microbial electrode.
2. Description of the Prior Art
In an industrial fermentation ramification such as L-amino acid fermentation and SCP fermentation, a feeding method by which a carbon source is continuously fed into the culture medium so that the concentration of the carbon source in the medium is controlled within a desired concentration during the fermentation is widely applied to obtain increased production.
In order to precisely control the concentration of a carbon source, on-line measurement of the carbon source in the culture medium is necessary, therefore a method for rapid, simple and quantitative determination of a carbon source is required.
Similarly, a method that is rapid, simple and quantitative in determining a fermentation product such as L-glutamic acid in the culture medium is also very effective for controlling the fermentation process, since the conclusion time of the fermentation can be easily decided.
According to a conventional method it is possible to determine non volatile carbon sources such as glucose and sucrose using an enzyme such as invertase and glucose oxidase together with a coloring material, or using an enzyme-electrode method comprising an electrode and immobilized enzyme. However, these conventional methods are not adapted for use in the determination of non-volatile carbon sources in a culture medium, when such a culture medium especially contains cane or beet molasses as a carbon source as well as containing a number of other organic compounds. Therefore, in practice, a conventional method for determining the reducing power of a saccharide is still employed. However, this common method is not satisfactory for the purpose of on-line measurements, since this common method is largely effected by reducing impurities usually coexisting in a culture medium during fermentation. Moreover, it requires complicated hydrolysis steps of the carbon source if the carbon source used is not a monosaccharide.
Accordingly, by use of a common gas chromatography or a semiconductor sensor, volatile carbon sources such as methanol, ethanol and acetic acid may be determined. These methods however are also unsatisfactory for on-line measurements, since a large amount of non-volatile organic impurities contained in a culture medium spoil the column of the gas-chromotography apparatus making long-term determination impossible. Further, this method is too unstable for the semiconductor sensor to be applied for on-line measurements.
On the other hand, it is possible to determine a fermentation product such as an L-amino acid according to the conventional Warburg method or the auto-analyzer method. These determine manometrically or colorimetrically the amount produced by the decarboxylation reaction (I) catalyzed by an L-amino acid decarboxylase such as L-glutamic acid or L-lysine decarboxylase derived from pumpkin, Esherichia coli or Bacterium cadaveris. ##EQU1##
These common methods are excellent in accuracy but not economical since an expensive enzyme has to be used batchwise and cannot be used continuously, and a very expensive determination apparatus is required for the auto-analyzer method. Furthermore, in all cases a complicated filtration process is necessary to remove microbial cells from the culture medium prior to the determination.